Recent update in diagnosis and treatment of human …ICT, Immunochromatographic test; ELISA, Enzyme-linked immunosorbent assay; SSKI, Saturated solu-tion of potassium iodide. Full-size

Submitted 11 October 2019Accepted 13 January 2020Published 20 February 2020

Corresponding authorsKaevalin Lekhanont,[email protected] Krajaejun,[email protected]

Academic editorErika Braga

Additional Information andDeclarations can be found onpage 20

DOI 10.7717/peerj.8555

Copyright2020 Chitasombat et al.

Distributed underCreative Commons CC-BY 4.0

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Recent update in diagnosis and treatmentof human pythiosisMaria Nina Chitasombat1,*, Passara Jongkhajornpong2,*, Kaevalin Lekhanont2

and Theerapong Krajaejun3

1Division of Infectious Disease, Department of Medicine, Faculty of Medicine, Ramathibodi Hospital,Mahidol University, Bangkok, Thailand

2Department of Ophthalmology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok,Thailand

3Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok,Thailand

*These authors contributed equally to this work.

ABSTRACTHuman pythiosis is an infectious condition with high morbidity and mortality. Thecausative agent is the oomycete microorganism Pythium insidiosum. The pathogeninhabits ubiquitously in a wet environment, and direct exposure to the pathogeninitiates the infection. Most patients with pythiosis require surgical removal of theaffected organ, and many patients die from the disease. Awareness of pythiosisamong healthcare personnel is increasing. In this review, we summarized and updatedinformation on the diagnosis and treatment of human pythiosis. Vascular and ocularpythiosis are common clinical manifestations. Recognition of the typical clinicalfeatures of pythiosis is essential for early diagnosis. The definitive diagnosis of thedisease requires laboratory testing, such as microbiological, serological, molecular, andproteomic assays. In vascular pythiosis, surgical intervention to achieve the organism-freemargin of the affected tissue, in combination with the use of antifungal drugs and P.insidiosum immunotherapy, remains the recommended treatment. Ocular pythiosis isa serious condition and earliest therapeutic penetrating keratoplasty with wide surgicalmargin is the mainstay treatment. Thorough clinical assessment is essential in allpatients to evaluate the treatment response and detect an early sign of the diseaserecurrence. In conclusion, early diagnosis and proper management are the keys to anoptimal outcome of the patients with pythiosis.

Subjects Mycology, Infectious Diseases, OphthalmologyKeywords Pythium insidiosum, Pythiosis, Diagnosis, Treatment, Management

INTRODUCTIONHuman pythiosis is a life-threatening infectious condition exhibiting high morbidity andmortality, as most patients require surgical removal of the affected organ (Thianprasit,Chaiprasert & Imwidthaya, 1996;Krajaejun et al., 2006b;Gaastra et al., 2010). The causativeagent, Pythium insidiosum, is a member of the unique group of aquatic fungus-likemicroorganisms, called oomycetes (De Cock et al., 1987), which is a common inhabitantof wet environments (Supabandhu et al., 2008; Vanittanakom et al., 2014; Presser & Goss,2015). Direct exposure to its infectious form, zoospore, can initiate infection (Mendoza,

How to cite this article Chitasombat MN, Jongkhajornpong P, Lekhanont K, Krajaejun T. 2020. Recent update in diagnosis and treat-ment of human pythiosis. PeerJ 8:e8555 http://doi.org/10.7717/peerj.8555

Hernandez & Ajello, 1993). With an increase in incidence, especially from the tropical,subtropical, and temperate countries, such as Thailand (Krajaejun et al., 2006b; Lekhanontet al., 2009; Permpalung et al., 2015; Lelievre et al., 2015; Anutarapongpan et al., 2018;Worasilchai et al., 2018; Permpalung et al., 2019), India (Bagga et al., 2018; Chatterjee& Agrawal, 2018; Rathi et al., 2018; Hasika et al., 2019; Agarwal et al., 2019; Appavu,Prajna & Rajapandian, 2019), Malaysia (Badenoch et al., 2001), China (He et al., 2016),Japan (Maeno et al., 2019), Australia (Badenoch et al., 2009), New Zealand (Murdoch &Parr, 1997), Spain (Bernheim et al., 2019), Israel (Tanhehco et al., 2011; Barequet, Lavinsky& Rosner, 2013), Columbia (Ros Castellar et al., 2017), Brazil (Bosco Sde et al., 2005), CostaRica (Neufeld et al., 2018), Jamaica (Pan et al., 2014), and the United States (Shenep et al.,1998), there has been an increase in awareness of pythiosis among healthcare personnel.

The major challenges in clinical management of pythiosis are the unavailability ofestablished diagnostic tests and effective therapeutic modalities. Prompt diagnosis andproper treatment are critical for ensuring a more favorable prognosis. In this review, wecompiled recent information on the diagnosis and treatment of human pythiosis, reportedin the literature by the healthcare professions who share their in-depth experiences of thedisease.

SURVEY METHODOLOGYThis study was approved by the Committee for Research, Faculty of Medicine RamathibodiHospital,Mahidol University (approval number:MURA2019/740). Our team, as healthcareprofessions in the fields of infectious diseases, ophthalmology, and pathology, performeda literature search using the PubMed database (https://www.ncbi.nlm.nih.gov/pubmed/).Based on the keywords, ‘pythiosis’ and ‘Pythium insidiosum’ articles extracted weresummarized to identify clinical information that was relevant to diagnosis andmanagementof human pythiosis and grouped into sections (i.e., clinical features, predisposing factors,laboratory diagnosis, management and follow-up, and clinical outcomes) and key notes(Fig. 1).

CLINICAL FEATURES OF PYTHIOSISHuman pythiosis exhibits heterogenous clinical manifestations (Krajaejun et al., 2006b;Sathapatayavongs et al., 1989). Most patients present with symptoms and signs that areassociated with the P. insidiosum infection of the medium–large artery (called vascularpythiosis) and the eye (called ocular pythiosis) (Krajaejun et al., 2006b). Unusual features ofthe disease include necrotizing cellulitis, deep neck abscesses, carotid aneurysm,meningitis,brain abscesses, cerebral septic emboli, and disseminated infection (Krajaejun et al., 2006b;Chitasombat et al., 2018a).

Vascular pythiosisVascular pythiosis usually affects the arteries of lower extremities (Fig. 2). Skin is likelya portal of entry where the zoospore of P. insidiosum attaches and germinates to invadearteries and surrounding tissue. Typical patients have an underlying hematological disorder

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Figure 1 Essential information for the diagnosis andmanagement of human pythiosis. Informationon host factor (A), environmental factor (B), and clinical presentation (C) provide clinical clues of pythio-sis. Once pythiosis is suspected, definitive diagnosis and proper treatment are urgent and essential for con-trolling infection (D). During treatment, clinical assessment of the patient with pythiosis should be per-formed daily (E). Abbreviations: GMS; Gomori-Methenamine Silver stain; IHC, immunohistochemistry;ICT, Immunochromatographic test; ELISA, Enzyme-linked immunosorbent assay; SSKI, Saturated solu-tion of potassium iodide.

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and an agriculture-related occupation. Most patients present with chronic non-healingskin lesions, arterial insufficiency syndrome (i.e., intermittent claudication, paresthesia,absence of arterial pulse, and evidence of arteritis or thrombosis), gangrenous ulcer, andpulsatile mass (i.e., a groin mass of the iliac or femoral aneurysm, and an abdominalmass of aortic aneurysm) (Krajaejun et al., 2006b; Permpalung et al., 2015; Reanpang etal., 2015; Sermsathanasawadi et al., 2016). Skin changes (i.e., color and texture), as thesequelae of arterial insufficiency, can be observed in patients with vascular pythiosis. The

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Figure 2 Clinical features of vascular pythiosis. (A) Gangrenous ulcer of the right foot (arrow); (B) Pe-ripheral run-off computerized tomography angiography shows total occlusion of the right proximal-to-distal popliteal artery (arrow). (Photo credit: Patawee Boontanondha, M.D.).

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infection usually ascends to a proximal arterial issue, which leads to thrombosis, arterialocclusion, and aneurysm (ruptured aneurysm results in mortality). Vascular pythiosis ofthe upper extremities has been occasionally reported (Worasilchai et al., 2018; Khunkhet,Rattanakaemakorn & Rajatanavin, 2015). There are only a few cases of carotid arteryinvolvement, an extremely rare but potentially fatal condition that results in meningitis,cerebral septic emboli, brain abscesses, and death. Patients with an immunosuppressed orneutropenic condition may present with abrupt-onset infection (i.e., within days after theonset of symptoms), with or without a history of exposure to aquatic habitats (Chitasombatet al., 2018a;Hoffman, Cornish & Simonsen, 2011;Hilton et al., 2016), and develop necroticskin lesions/cellulitis that progress to vascular infections shortly afterward (within a fewdays or weeks).

Early recognition of pythiosis and the use of a rapid laboratory test leads to promptand definitive therapy. Laboratory investigation (see the ‘‘Laboratory diagnosis’’ section) isessential for the identification of the P. insidiosum infection. Specimen handling is a criticalstep to enhance the success rate of the laboratory investigation. Specimens such as bloodclot, pus, and vessel tissue (at the site of thrombosed vessels and proximal margin), shouldbe sent, without formalin preservation, to a clinical microbiology laboratory for cultureidentification, staining, and/or molecular testing. Resected vessel and soft tissue specimensent for histological examination should be labelled—proximal or distal—in order toidentify the disease-free surgical margin by Grocott-Gömöri methenamine silver (GMS)

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staining (Sermsathanasawadi et al., 2016). Further immunohistochemistry staining for P.insidiosum is helpful to confirm the diagnosis, especially if the standard microbiologicalculture is not available or fails to identify the pathogen (Inkomlue et al., 2016; Keeratijarutet al., 2009).

Ocular pythiosisPatients with ocular pythiosis (also known as Pythium keratitis) usually have a history ofpredisposing factors (see the ‘‘Predisposing factors’’ section), and present with ocular pain,irritation, photophobia, decreased visual acuity, conjunctival redness, and eyelid swelling,similar to another microbial keratitis. Clinical onset of ocular pythiosis could range fromtwo days to over a month. Pythium keratitis is often clinically indistinguishable from fungalkeratitis, because the causative agents share some clinical features (such as, the presence ofgrayish-white stromal infiltrates with feathery margins on slit-lamp biomicroscopy) andmicroscopic findings (such as, septate linear branching structures in a corneal scrapingsample). However, the natural history and clinical clues at the margin or surrounding areasof the main infiltrates would help to differentiate Pythium keratitis from fungal keratitis.

Patients with Pythium keratitis usually present with typical clinical manifestations,includingmultiple, linear, tentacle-like infiltrates anddot-like or pinhead-shaped infiltrates,involving the subepithelial, anterior stromal, and midstromal layers in surrounding corneaand radiating in a reticular pattern from the central area of the lesion towards the limbus(Fig. 3) (Lekhanont et al., 2009; Lelievre et al., 2015; Bagga et al., 2018;Chatterjee & Agrawal,2018; Agarwal et al., 2019; He et al., 2016; Thanathanee et al., 2013; Sharma et al., 2015;Agarwal et al., 2018). Radial keratoneuritis has been observed in some cases (Lekhanontet al., 2009; He et al., 2016). Corneal lesions should be closely monitored, as the typicalfeatures of Pythium keratitis may develop during the course of the disease (Chatterjee& Agrawal, 2018). In advance disease, these clinical clues may be obscured by densecorneal stromal infiltration (Anutarapongpan et al., 2018; Badenoch et al., 2001). The rapidand progressive nature of ocular pythiosis, despite intensive antimicrobial treatment,distinguishes Pythium keratitis from other fungal infections. An extensive and aggressiveinfiltration of P . insidiosum could result in corneal perforation, anterior chamber, limbaland scleral invasions, or endophthalmitis in a few days or weeks (Lekhanont et al., 2009;Lelievre et al., 2015; Rathi et al., 2018; Agarwal et al., 2019; Badenoch et al., 2001; He et al.,2016; Maeno et al., 2019; Ros Castellar et al., 2017; Thanathanee et al., 2013; Sharma et al.,2015; Agarwal et al., 2018).

Corneal scrapings should be subjected to clinical microbiology examination (i.e., fungalculture, polymerase chain reaction (PCR), and DNA sequencing; see the ‘‘Laboratorydiagnosis’’ section) to establish a definitive diagnosis. An ocular specimen is usuallyobtained in a minimal amount, which could lead to a false-negative result by suchlaboratory tests. Serological tests are not useful because the serum anti-P. insidiosumantibody in patients with Pythium keratitis is usually undetectable (Krajaejun et al., 2006b;Permpalung et al., 2019; Krajaejun et al., 2009; Jindayok et al., 2009). Repeated cornealcultures or combining specimens, including corneal scrapings and corneal buttons, couldincrease the chance of a positive result (Hasika et al., 2019).

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Figure 3 Clinical features of ocular pythiosis. Slit-lamp photograph of the infected cornea demonstratescentral, dense, grayish-white infiltrates with tentacle-like extensions (arrowhead) and subepithelial dot-like infiltrates radiating in a reticular pattern from the lesion (arrow). (Photo credit: Passara Jongkhajorn-pong, M.D.).

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In vivo confocal microscopy (IVCM) is an emerging, non-invasive, real-time imagingtechnique that enables morphological and quantitative analysis of ocular surfacemicrostructure in both health and disease. Clinically, IVCM is a useful diagnostic tool forthe early diagnosis of microbial particularly fungal and Acanthamoeba keratitis (Alzubaidiet al., 2016). P. insidiosum hyphae can be identified using IVCM in 95% of culture-or PCR-positive patients (Anutarapongpan et al., 2018). Pythium keratitis manifest ashyperreflective branching structures (vary in size; 1.5–7.5 µm in diameter and 90-400µm in length), septate linear branching structures resembling fungal filaments, andirregular filaments arranged in a sinuous, various branching pattern, forming ‘‘X’’,’’Y’’, and’’ S’’ shapes as seen by IVCM (Lelievre et al., 2015; He et al., 2016; Tanhehcoet al., 2011; Thanathanee et al., 2013). However, microbial characteristics captured byIVCM are non-specific and overlapped among Pythium, Aspergillus, and Fusariumkeratitis (Anutarapongpan et al., 2018). Nevertheless, IVCM can provide rapid visualizationof Pythium hyphae in infected cornea and is recommended when Pythium keratitis issuspected (Anutarapongpan et al., 2018).

Unusual features of pythiosisPatients with pythiosis can present with various skin manifestations, such as vesicle/bulla,skin ulcer, cellulitis, chronic swelling, painful subcutaneous lesion, infiltrative lump andulcer on the limb (Krajaejun et al., 2006b) and rapidly progressive P. insidiosum infection(i.e., necrotizing cellulitis) (Krajaejun et al., 2006b; Kirzhner et al., 2015). If left untreated,cutaneous/subcutaneous infection could progress to vascular pythiosis, which can lead toamputation of the affected limb (Krajaejun et al., 2006b; Khunkhet, Rattanakaemakorn &

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Rajatanavin, 2015). Craniofacial infection, such as periorbital/orbital necrotizing cellulitis,is a rapid-onset clinical feature observed in children and young adults with a recent historyof exposure to wetland (Krajaejun et al., 2006b; Hilton et al., 2016; Kirzhner et al., 2015;Triscott, Weedon & Cabana, 1993). Rarely, P. insidiosum infection can disseminate to avisceral organ and result in mortality (Krajaejun et al., 2006b), which in a patient withgastrointestinal involvement leads to upper gastrointestinal bleeding, bloody mucousstools, gastric/ileal ulcer, and peritonitis (Krajaejun et al., 2006b).

PREDISPOSING FACTORSHost (intrinsic) factorsVascular pythiosis has been strongly associated with thalassemia (Krajaejun et al., 2006a;Permpalung et al., 2015; Worasilchai et al., 2018; Reanpang et al., 2015; Sermsathanasawadiet al., 2016; Thitithanyanont et al., 1998). When stimulate with P. insidiosum zoospore,peripheral blood mononuclear cells collected from thalassemic patients showed lowerlevel of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferongamma (IFN-γ) production, compared with the non-thalassemic controls (Ud-Naen etal., 2019). Other hematologic disorders that predispose individuals to vascular pythiosisinclude paroxysmal nocturnal hemoglobinuria, aplastic anemia, myelodysplasia, idiopathicthrombocytopenic purpura, and leukemia (Krajaejun et al., 2006b), and to a lesser extent,young age, alcoholism, malnourishment, immunosuppression, HIV infection, cancer, andneutropenia (Krajaejun et al., 2006b; Pan et al., 2014; Shenep et al., 1998;Chitasombat et al.,2018a;Hoffman, Cornish & Simonsen, 2011;Hilton et al., 2016; Kirzhner et al., 2015; Francoet al., 2010; Chitasombat et al., 2018b).

In contrast to vascular pythiosis, ocular pythiosis commonly affects healthyindividuals (Krajaejun et al., 2006b; Lekhanont et al., 2009; Krajaejun et al., 2004).However, in a series reported from Northeastern Thailand, 50% of patients with Pythiumkeratitis had thalassemia hemoglobinopathy (Thanathanee et al., 2013), though theprevalence, and the heterogeneity, of thalassemia hemoglobinopathies is high amongThai people living especially in Northeastern Thailand. Thus, whether thalassemiais a predisposing factor in ocular pythiosis is not known, warranting the need foradditional studies to understand the relationship between ocular pythiosis and thalassemiahemoglobinopathies. Additionally, a fulminant form of Pythium keratitis can be observedin immunocompromised patients (such as those with diabetes or Crohn’s disease treatedwith immunotherapy), which resulted in poor clinical outcomes (i.e., enucleation) (Rathiet al., 2018; Neufeld et al., 2018).

Environmental (extrinsic) factorsP. insidiosum inhabits stagnant water and soil, and colonizes aquatic vegetation (Su-pabandhu et al., 2008; Vanittanakom et al., 2014; Mendoza, Hernandez & Ajello, 1993).Potential risk factors for pythiosis include farming exposure, direct contact with a waterresource (i.e., lake, river, lagoon, swamp, or even swimming pool), and a history oftravel to endemic countries, such as Thailand (Krajaejun et al., 2006b; Lelievre et al.,2015; Hasika et al., 2019; Appavu, Prajna & Rajapandian, 2019). Inappropriate hygiene

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of contact lens can pose a risk (Lekhanont et al., 2009; Lelievre et al., 2015; Badenoch et al.,2001; Maeno et al., 2019; Bernheim et al., 2019; Tanhehco et al., 2011; Barequet, Lavinsky& Rosner, 2013; Neufeld et al., 2018; Raghavan et al., 2018), and exposure to dust or someforeign body in their eyes (Bagga et al., 2018; Hasika et al., 2019) predisposes individualswith no agricultural activity to this infection. The incidence of pythiosis follows a seasonaltrend (Hasika et al., 2019; Thanathanee et al., 2013) with outbreaks of Pythium keratitisoccurring during the rainy season in Thailand (Thanathanee et al., 2013) and around Juneto September during monsoon season in South India (Hasika et al., 2019).

LABORATORY DIAGNOSISDiagnosis of pythiosis is necessary to ensure prompt treatment and promote betterprognosis of pythiosis patients. This requires diligent records of medical history, physicalexamination of the patient, and laboratory information for diagnosing definitive pythiosisusing laboratory methods summarized below:

Direct examinationClinical specimens (i.e., purulent discharge, corneal scraping, blood clot, and pathologictissue) obtained from a lesion are subjected to direct wet mount examination, using 10%KOH preparation (Krajaejun et al., 2006b; Mendoza, Ajello & McGinnis, 1996). Althoughthe results obtainedmay not be specific, microscopic observation of broad and occasionallysparsely septate filamentous elements alerts to a further identification of the causativeorganism (i.e., P. insidiosum and some other pathogenic fungi).

Sample handling and culture identificationTransportation of an infected patient tissue with suspected pythiosis to a clinicalmicrobiology laboratory is a critical step for successful isolation of P. insidiosum.Temperature control is essential to ensure viability, and failure to isolate P. insidiosumis linked to storage of clinical specimen in low-temperature containers, such as icebox,refrigerator, or freezer (Brown & Roberts, 1988; Bentinck-Smith et al., 1989). Optimalgrowth of P. insidiosum occurs between 28 ◦C and 37 ◦C and extreme temperature as lowas 8 ◦C or as high as 42 ◦C can completely inhibit or even kill the organism (Krajaejunet al., 2010). When immediate transportation of a culture specimen is not possible, it isrecommended to store the sample in sterile distilled water while transferring to the clinicalmicrobiology laboratory (Mendoza, Ajello & McGinnis, 1996).

P. insidiosum grows well on standard agar types, such as Sabouraud dextrose agar,potato dextrose agar, corn meal agar, and blood agar (Chaiprasert et al., 1990; Grooters etal., 2002). After inoculation of a viable P. insidiosum-containing tissue on agar of choice,a yellow-to-white or colorless-to-white flat colony can be observed within a few days(the color may depend on the culture medium used and the age of the colony), with aradial growth rate of ∼5 mm/day (De Cock et al., 1987; Krajaejun et al., 2010). Under themicroscope, P. insidiosum appears as a right-angle branching, sparsely septate, broad (upto 10 µm in diameter) hyphae (Mendoza, Ajello & McGinnis, 1996). Zoosporangium (a sacformed at the hyphal tip that contains multiple mobile biflagellate zoospores) is a unique

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characteristic that differentiates P. insidiosum from fungi. Zoosporogenesis can be inducedin an experienced laboratory by using sterile grass leaves and the induction solution,as described in details elsewhere (Chaiprasert et al., 1990; Mendoza & Prendas, 1988).Although P. insidiosum is a prominent pathogenic oomycete of humans, other oomycetes,such as Lagenidium spp. and Pythium aphanidermatum have also been reported, to a muchlesser extent, as a human pathogen (Calvano et al., 2011; Reinprayoon et al., 2013; Farmer etal., 2015). Of late, the accurate identification of P. insidiosum requires immunodiagnostic,molecular, or proteomic assay (Badenoch et al., 2001; Vanittanakom et al., 2004; Intaramatet al., 2016; Krajaejun et al., 2018).

Serological tests and biomarkersDetection of anti-P. insidiosum antibody can facilitate the diagnosis of pythiosis. Severalmethods have been developed to detect serum P. insidiosum-specific antibodies. Twotypes of crude proteins, (i) culture filtrate antigen (CFA) and (ii) soluble antigen frombroken hyphae (SABH) (Krajaejun et al., 2006a), extracted from P. insidiosum serve asantigens for serological tests. Immunodiffusion (ID) is a simple, cost-effective, andspecific assay that is designed to observe in-gel immunoprecipitation lines generatedby anti-P. insidiosum antibodies in patient serum and the P. insidiosum crude proteinextract (Imwidthaya & Srimuang, 1989; Pracharktam et al., 1991). Major drawbacks ofID include poor detection sensitivity leading to a false-negative result, and longerturnaround time (∼24 h) (Krajaejun et al., 2009; Jindayok et al., 2009; Krajaejun et al.,2002; Chareonsirisuthigul et al., 2013). An enzyme-linked immunosorbent assay (ELISA)has circumvented the limitation of ID, as it shows higher sensitivity and shorter turnaroundtime (∼3–4 h) (Intaramat et al., 2016; Krajaejun et al., 2002; Chareonsirisuthigul et al.,2013). Western blot (WB) for immunodetection of P. insidiosum (Krajaejun et al., 2006a;Supabandhu et al., 2009) has been developed though its diagnostic application is limiteddue to the multi-step procedures involved and complexity. Hemagglutination (HA)and immunochromatographic test (ICT) are rapid and user-friendly assays that detectanti-P. insidiosum antibodies within 30–60 min (Krajaejun et al., 2009; Jindayok et al.,2009; Intaramat et al., 2016). However, implementation of a serodiagnostic test in a non-endemic region is challenging. Transportation of the specimen could impact the assayturnaround time.

Comparison of the above-mentioned serological assays, using a set of 37 pythiosispatients and 248 control sera, shows that ELISA (sensitivity, 100%; specificity, 100%) andICT (sensitivity, 100%; specificity, 100%) outperform ID (sensitivity, 68%; specificity,100%) and HA (sensitivity, 84%; specificity, 82%) (Chareonsirisuthigul et al., 2013).Evaluation of ELISA and ICT using a different set of 28 pythiosis and 80 control sera,demonstrates the greater sensitivity of ELISA compared with ICT (96% vs. 86%) (Intaramatet al., 2016). Each serological assay has its own advantages and disadvantages. Selection ofan appropriate test for serodiagnosis of pythiosis depends on assay availability, experienceof the laboratory personnel, preferred test performance and turnaround time. Mostimportantly, the serological assays described here often fail to detect anti-P. insidiosumantibodies in patients with ocular pythiosis (Krajaejun et al., 2009; Jindayok et al., 2009;

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Intaramat et al., 2016). Therefore, one should be aware of false-negative results whenperforming the assays on samples from an ocular patient.

During the clinical course of pythiosis, levels of anti-P . insidiosum antibodies canbe monitored using one of the serological tests. A gradual decrease in anti-P. insidiosumantibody levels over the course ofmonths, in associationwith clinical improvement, indicatefavorable prognosis (Jindayok et al., 2009; Pracharktam et al., 1991; Krajaejun et al., 2002).In contrast, increased or sustained antibody levels may be observed after administrationof the P. insidiosum immunotherapeutic antigen (PIA) vaccine, prepared from crudeprotein extracts of the pathogen, in clinically-cured pythiosis patients (Worasilchai etal., 2018; Krajaejun et al., 2002). Recently, Worasilchai et al. (2018) reported the use ofserum β-d-glucan as a surrogate marker for monitoring 50 vascular pythiosis patientsafter a combination of medical and surgical treatment. A decrease in β-d-glucan levels wasobserved over a few months in 45 patients who survived, in contrast to the high level ofβ-d-glucan in five patients who died. However, the serum β-d-glucan was not specific toP. insidiosum and therefore, all possible causes that affect β-d-glucan level should be ruledout to better understand the role of β-d-glucan.

Histological examinationStandard histological stains, such as GMS and Periodic acid-Schiff (PAS) demonstrate thepresence and extent of P. insidiosum in infected tissues (Krajaejun et al., 2006b; Keeratijarutet al., 2009; Mendoza, Prasla & Ajello, 2004). However, such stains may not differentiate P.insidiosum from other fungi (i.e., Aspergillus spp., Fusarium spp., Mucorales). Hematoxylinand eosin (H&E) staining shows tissue reactions, such as infiltration of eosinophils,purulent granuloma, and giant cells at the P. insidiosum infection site (Krajaejun et al.,2006b).

As opposed to the standard stains (i.e., GMS, PAS andH&E), several immunohistologicalstaining assays have been developed to facilitate microscopic detection of P.insidiosum (Inkomlue et al., 2016; Keeratijarut et al., 2009; Triscott, Weedon & Cabana,1993; Mendoza, Ajello & McGinnis, 1996; Mendoza, Kaufman & Standard, 1987; Brownet al., 1988). These assays rely on the use of rabbit antiserum generated against crudeprotein extract of P. insidiosum. For example, Mendoza et al. implemented a directimmunofluorescent assay using the rabbit anti-P. insidiosum antibodies conjugatedwith fluorescein isothiocyanate (Mendoza, Ajello & McGinnis, 1996; Mendoza, Kaufman &Standard, 1987). Brown et al. (1988), Triscott, Weedon & Cabana (1993), and Keeratijarutet al. (2009) introduced indirect immunochemical assays that require sequential stainingreactions with rabbit anti-P. insidiosum antibodies (primary antibody), mouse or bovineanti-rabbit IgG (secondary antibody) tagged with peroxidase, and enzymatic substrates.These immunostaining assays require careful examination under a light microscope toconfirm the presence of P. insidiosum in infected tissues. Interpretation of results may becomplicated by false-positive staining of other fungi, such as Conidiobolus and Fusariumspecies (Keeratijarut et al., 2009; Grooters & Gee, 2002). Thus, confirmation of the P.insidiosum infection requires additional laboratory investigations. When fungal elementsare histologically detected by GMS or PAS, a negative immunostaining result could exclude

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the possibility of P. insidiosum, due to the high sensitivity and negative predictive value ofthe tests (Keeratijarut et al., 2009).

A novel immunohistological method targeting elicitins that are only present inoomycetes, including P. insidiosum (Jiang et al., 2006; Lerksuthirat et al., 2015), hasbeen reported to be highly specific. Recently, Inkomlue et al. used a rabbit antiserumagainst recombinant elicitin (ELI025) of P. insidiosum for the development of animmunohistochemical assay (Inkomlue et al., 2016), and found that all 38 P. insidiosumsamples were detected, but not 49 control samples of various fungi, including Fusariumspecies and Mucorales.

Molecular analysisConventional molecular techniques used to detect P. insidiosum are sequence homologyanalysis and PCR. The most popular target sequence is rDNA (also known as ribosomalRNA gene repeat). The rDNA contains 18S ribosomal RNA, internal transcribed spacer1, 5.8S ribosomal RNA, internal transcribed spacer 2, and 28S ribosomal RNA (Grooters& Gee, 2002). These molecular techniques require a combination of universal fungal- orP. insidiosum-specific primers, template DNA extracted from pure culture or infectedtissue, PCR reagents and equipment, and DNA sequencing facility (Badenoch et al., 2001;Vanittanakom et al., 2004; Znajda, Grooters & Marsella, 2002). Use of molecular techniquescan substitute microbiological and immunological methods, especially when the latter failto detect P. insidiosum. Badenoch et al. used universal fungal primers to amplify andsequence a DNA fragment from an organism isolated from a patient with an unknownocular infection (Badenoch et al., 2001). Homology analysis of the obtained sequence (byBLAST searching against the GenBank database) identified the organism as P. insidiosum.Although the sequence homology analysis is a time-consuming and multi-step procedure,it is a standard method for the identification of many microorganisms, including P.insidiosum. One reason is that most reagents and tools required for this test are readilyavailable in many clinical microbiology laboratories.

Using species-specific primers, PCR amplification can identify P. insidiosum from pureculture or infected tissue without DNA sequencing of the amplicon (Vanittanakom et al.,2004; Grooters & Gee, 2002; Znajda, Grooters & Marsella, 2002). Compared to sequencehomology analysis, the PCR assay significantly reduces turnaround time from days tohours. Grooters et al. and Znajda et al. have co-developed a nested PCR assay, usingthe universal fungal primers (ITS1 and ITS2 or ITS2P) for the first-round reaction andthe species-specific primers (PI-1 and PI-2) for the second-round reaction to specificallyamplify P. insidiosum DNA (Grooters & Gee, 2002; Znajda, Grooters & Marsella, 2002).Vanittanakom et al. showed that the primers PI-1 and PI-2 (Grooters & Gee, 2002) failedto detect a Thai P. insidiosum strain. Therefore, a new set of P. insidiosum-specific primers(ITSpy1 and ITSpy2) was used in a simplified one round-PCR reaction (Vanittanakomet al., 2004) that was able to detect all four Thai strains tested. Thongsri et al. reported asingle-tube nested PCR using a different set of primers (CPL6, CPR8, YTL1, and YTR1)for specific detection of P. insidiosum (Thongsri et al., 2013). More recently, Rujirawat etal. developed a multiplex PCR assay, using four primers (ITS1, Ra, R2, and R3) to target

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several single nucleotide polymorphisms of rDNA (Rujirawat et al., 2017). The multiplexPCR assay can simultaneously identify and genotype P. insidiosum.

Keeratijarut et al. showed that the rDNA-targeting primers, ITSpy1 and ITSpy2,were unable to PCR amplify certain P. insidiosum strains (Keeratijarut et al., 2014). Theyproposed the exo-1,3- β-glucanase-encoding gene (exo 1) of P. insidiosum as an alternativePCR target, and designed exo 1-specific primers,Dx3 andDx4, for a conventional PCRassay.Based on a head-to-head comparison for PCR-based detection of 35 Thai P. insidiosumstrains, the primers, Dx3 and Dx4, showed higher detection sensitivity than with ITSpy1and ITSpy2 (100% vs. 91%) (Keeratijarut et al., 2014). Besides, the primers, Dx3 and Dx4,did not amplify non-specific target DNA from a variety of fungal species tested, showing100% detection specificity. Keeratijarut et al. introduced an exo 1-targeting real-time PCRassay using the primers, Pr77 and Pr78, for detection of P. insidiosum (Keeratijarut et al.,2015). The detection sensitivity (100%) and specificity (100%) of the real-time PCR washigh (Keeratijarut et al., 2015), but shortened the assay turnaround time by eliminatinglaborious and toxic steps (i.e., gel electrophoresis and ethidium bromide staining).

Proteomic analysisMatrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOFMS) is a powerful diagnostic tool (Singhal et al., 2015; Sanguinetti & Posteraro, 2017;Jang & Kim, 2018) that can be applied for accurate identification of clinically-importantmicroorganisms at low cost and short turnaround time. Unlike serological and molecularassays, MALDI-TOF MS does not require pathogen-specific reagents, such as primer,antigen, and antibody. Crude proteins of an unknown microorganism are extracted usingan optimized protocol and subjected to the generation of main spectral profile (MSP)by MALDI-TOF MS analysis. The obtained MSP is searched against a reference databasecontaining MSPs of many typed microorganisms. The matched organism with a significantscore is, therefore, the identity of the unknown sample. Large mass spectrometric databasesof a wide variety of microorganisms are currently available and accessible to assist theMALDI-TOF MS-based microbial identification (Singhal et al., 2015; Lau et al., 2013;Becker et al., 2014). Several mass spectrometric databases are now supplemented withthe P. insidiosum MSPs aiding the identification of P. insidiosum (Bernheim et al., 2019;Krajaejun et al., 2018; Mani et al., 2019). The microorganism in question is reported as P.insidiosum if the MSP generated significantly matched its corresponding mass spectrumdeposited in the reference database.

MANAGEMENT AND FOLLOW-UPThe treatment of pythiosis can be categorized into surgical intervention, medications(antimicrobial and related agents), and immunotherapy. Based on the experiences of ourcenter and that of other groups, P. insidiosum is usually resistant to both systemic andtraditional topical antifungal agents. Since aggressive medical treatment often fails to curethe infection, prompt surgical intervention is the key treatment to control the disease.Potential therapeutic options for patients with vascular and ocular pythiosis are presentedbelow:

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Vascular pythiosisPresurgical assessmentUrgent preoperative assessment determined by computerized tomography angiogram(CTA) of affected vessel (i.e., lower extremities) up to the proximal origin of vessels (i.e.,CTA of whole aorta with femoral run off) is recommended. Abnormal radiography showingthickening of the vessel wall with enhancement, thrombosis, and aneurysmal dilation mayindicate arteritis (Krajaejun et al., 2006b; Sermsathanasawadi et al., 2016; Chitasombat etal., 2018b). The CTA method has some advantages compared with angiogram for thedetermination of soft tissue and lymph node involvement. Magnetic resonance imagingwith angiogram (MRI/MRA) is the preferred modality to assess cranial involvement,i.e., septic emboli, small abscesses, and cavernous sinus extensions (Rathi et al., 2018;Chitasombat et al., 2018b; Narkwiboonwong et al., 2011).

Surgical interventionPrompt radical surgery leading to amputation to achieve pathogen-free surgicalmargin is the mainstay of successful treatment of vascular pythiosis (Krajaejun et al.,2006b; Worasilchai et al., 2018; Sermsathanasawadi et al., 2016). The suggested adequateresected proximal margin is 5 cm above the site of the arterial lesion detected byCTA (Sermsathanasawadi et al., 2016; Sangruchi et al., 2013). Prior to surgery, scheduledarrangement with pathologist ensures immediate intraoperative results to enable surgicaldecisions. Intraoperative soft tissue and vascular margin should be assessed carefullyfor macroscopic (gross appearance) and microscopic examination with real-time frozensection for KOH stain (Sermsathanasawadi et al., 2016). Intraoperative frozen sectionspecimen is crucial to determine the free surgical margin (Chitasombat et al., 2018a;Sermsathanasawadi et al., 2016). The organism-positive margin required re-excision at≥ 5 cm proximally, and re-examination by KOH test until the arterial margins andsurrounding soft tissue margins were free of disease (Sermsathanasawadi et al., 2016).Inadequate assessment of surgical-free margin at the time of surgery leads to progression ofresidual disease involving the proximal artery, i.e., aorta (Chitasombat et al., 2018a). Somepatients require repeated aggressive surgical procedure, i.e., below/above knee amputation,hip disarticulation, hemipelvectomy, and aneurysmectomy (Permpalung et al., 2015).Patients with common iliac artery or aortic involvement who underwent aneurysmectomywith bypass grafting survived only few months (Krajaejun et al., 2006b; Permpalung et al.,2015; Sathapatayavongs et al., 1989; Sermsathanasawadi et al., 2016; Wanachiwanawin etal., 1993).

Antifungal drugsAntifungals are generally ineffective against P. insidiosum as they do not target enzymesin the ergosterol biosynthetic pathway (Lerksuthirat et al., 2017). This confers clinicalresistance to antifungals, which could only be effective at the difficult-to-achieveconcentrations (Lerksuthirat et al., 2017). In vitrominimal inhibitory concentration (MIC)data revealed amphotericin B had the highest MIC, followed in order by voriconazole,fluconazole, anidulafungin, caspofungin, itraconazole, and terbinafine (Permpalung etal., 2015). Combination of itraconazole and terbinafine was commonly used, owing to

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its ability to synergistically inhibit growth in vitro (Krajaejun et al., 2006b; Shenep et al.,1998; Kirzhner et al., 2015; Argenta et al., 2008). However, synergistic effects betweenitraconazole or voriconazole and terbinafine could not be demonstrated in Thai P.insidiosum isolates (Permpalung et al., 2015;Worasilchai et al., 2018).

Prolonged treatment with a combination of itraconazole and terbinafine successfullycured a few patients with unresectable disease (Krajaejun et al., 2006b; Shenep et al., 1998).The in vitro activity of various antifungals were explored; terbinafine combined withcaspofungin or fluconazole showed synergistic activity in Brazilian isolates (Cavalheiro etal., 2009). Susceptibility should be cautiously interpreted as there are several methodsof antifungal susceptibility determination without standardized assays and regionaldifferences exist for various strains/genotypes (Permpalung et al., 2015; Lerksuthirat etal., 2017; Schurko et al., 2003a; Schurko et al., 2003b). New triazoles, i.e., voriconazoleand posaconazole, have been used to successfully treat patients with residual unresectabledisease (Kirzhner et al., 2015). Recent in vitro data revealed that 12.5% of Thai P. insidiosumstrains had voriconazole MIC >4 mg/l, whereas approximately 70–80% of isolates haditraconazole MIC >1 mg/l (Permpalung et al., 2015; Worasilchai et al., 2018; Permpalunget al., 2019; Susaengrat et al., 2019). Regarding the duration of treatment, combination ofitraconazole and terbinafine treatment for 1–2 years has been recommended based on asuccessful outcome of unresectable vascular pythiosis (Worasilchai et al., 2018; Shenep etal., 1998; Sermsathanasawadi et al., 2016). However, treatment protocols should be tailoredto the individual patient’s needs according to clinical, laboratory data, and radiographicinformation (see ‘follow-up and monitoring’).

Antimicrobial agentsHistorically, saturated solution of potassium iodide (SSKI; one mL orally three times perday for up to three months) has been used to successfully treat localized skin/subcutaneousdisease (Krajaejun et al., 2006b; Imwidthaya, 1994). Several antibacterial drugs that inhibitprotein synthesis can inhibit the growth ofP. insidiosum (Brazilian strains) in vitro includingazithromycin, tigecycline, clarithromycin, linezolid, nitrofurantoin, chloramphenicol,quinupristin/dalfopristin, clindamycin, josamycin, miltefosine, sutezolid, retapamulin,tiamulin, and valnemulin (Loreto et al., 2011; Mahl et al., 2012; Jesus et al., 2014; Jesus etal., 2016; Itaqui et al., 2016; Loreto et al., 2019). Azithromycin demonstrated potent in vivoactivity in an experimental model of pythiosis (Jesus et al., 2016; Loreto et al., 2018). In arecent study, azithromycin, doxycycline, and clarithromycin combined with itraconazoleor voriconazole were administered as salvage therapy in two patients with relapsed vascularpythiosis (aortic aneurysm), in whom surgery cannot be performed (Susaengrat et al.,2019). Both patients responded well to combination of doxycycline and clarithromycin orazithromycin and survived up to 64 weeks of follow-up (Susaengrat et al., 2019). In vitrosusceptibility of P. insidiosum isolates revealed clarithromycin, azithromycin, minocycline,and doxycycline MICs of 0.5, 2, 2, and 4 mg/l, respectively. Furthermore, azithromycin orclarithromycin combined with either minocycline or doxycycline had in vitro synergisticeffect (Susaengrat et al., 2019).

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Iron chelatorsHuman pythiosis was first described in a thalassemia patient in 1989 (Sathapatayavongset al., 1989). Since then, vascular pythiosis mainly affects thalassemia patients and ironoverload has been associated with the pathogenesis of pythiosis (Krajaejun et al., 2006b).P. insidiosum possesses the gene encoding ferrochelatase, which indicates role of iron inthe pathogenesis (Krajaejun et al., 2011). Iron overload is known to alter T and B cellproliferation (Schaible & Kaufmann, 2004) because of which, patients with thalassemia aresusceptible to infection owing to impaired immune responses in monocytes/macrophagesand cytokine production (Ud-Naen et al., 2019; Wanachiwanawin et al., 1993). Ironchelation augments tumor necrosis factor alpha (TNF-α), GM-CSF and IFN-γ releasefrom monocytes/macrophages in thalassemia patients irrespective of ferritin levels (Ud-Naen et al., 2019). In vitro data showed that deferasirox directly damages P. insidiosumhyphae (Zanette et al., 2015). In the rabbit model of pythiosis, deferasirox exhibits animmuno-modulating effect which is similar to that of the immunotherapy (Zanette et al.,2015). Clinically, various iron chelators, i.e., deferiprone, deferasirox, and deferoxaminehave been used adjunctively in thalassemia patients to treat iron overload (Permpalunget al., 2015; Worasilchai et al., 2018). Iron chelation therapy did not appear to change thetreatment outcome in vascular pythiosis (Permpalung et al., 2015).

ImmunotherapyThis strategy was first used successfully in 1998 as an adjunct treatment in patientswith unresectable disease (Khunkhet, Rattanakaemakorn & Rajatanavin, 2015). The PIAvaccine, derived from endoplasmic and secretory antigens of the pathogen (Krajaejun etal., 2006b), shifts immune response from the T-helper 2 to T-helper 1 for cytotoxic killingof hyphae (Wanachiwanawin et al., 2004). The vaccine demonstrated an acceptable safetyprofile, but the efficacy remains inconclusive due to the small sample size (Permpalunget al., 2015; Wanachiwanawin et al., 2004). Only a few patients with inoperable diseasesurvived (Khunkhet, Rattanakaemakorn & Rajatanavin, 2015; Susaengrat et al., 2019).Adverse reaction to the vaccine included local swelling, redness, pruritus, minor rash,and regional lymphadenopathy (Permpalung et al., 2015; Wanachiwanawin et al., 2004). Asevere inflammatory reaction at the site of infection showing a massive periorbital/facialswelling resulting in respiratory distress was reported in a pediatric patient fromthe United States after the third dose of vaccine (Kirzhner et al., 2015). Nowadaysin Thailand, PIA vaccine is administered as an adjunct treatment (Krajaejun et al.,2006b; Worasilchai et al., 2018; Sermsathanasawadi et al., 2016). Various PIA preparationsderived from protein antigen prepared from P. insidiosum have been used (Permpalunget al., 2015; Sermsathanasawadi et al., 2016; Wanachiwanawin et al., 2004; Mendoza,Mandy & Glass, 2003). The vaccination schedules varies across different institutionsin Thailand (Permpalung et al., 2015; Sermsathanasawadi et al., 2016). The commonlyadministered schedule in Thailand is vaccine preparations of one milliliter of 2 mg/ml PIAgiven subcutaneously at diagnosis and at 0.5, 1, 1.5, 3, 6, and 12 months (Worasilchai et al.,2018). Host response to PIA vaccine showed inter-individual variations. The measurementof P. insidiosum-specific antibody titer by ELISA can be used to monitor host immune

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to PIA vaccine; patient with higher titer in the absence of active ongoing infection hadsurvival advantage (Worasilchai et al., 2018).

Follow-up and monitoringDaily physical assessment of soft tissue, surgical sites, lymphadenopathy and signs ofvascular insufficiency are critical after surgery. Stump abscesses, and myositis alongwith evidence of arterial insufficiency syndrome (arteritis, thrombosis, aneurysm,pulsatile mass) represent residual disease that necessitates investigation and aggressivemanagement (Chitasombat et al., 2018a). Neutropenic patients should be monitoredclosely due to rapid clinical deterioration, i.e., progression of cellulitis (Chitasombat etal., 2018a). Early postoperative CTA may show non-specific findings, i.e., swelling, andthrombosis of stump at the ligated vessel (Chitasombat et al., 2018a). Follow-up imagingshould be performed if disease relapse is suspected, which can occur several months aftersurgery (Susaengrat et al., 2019). Arteritis should be suspected when certain radiographicclues, such as thickening of the vessel wall with contrast enhancement, thrombosis, andaneurysmal dilation, are observed (Chitasombat et al., 2018a; Sermsathanasawadi et al.,2016). Patients with disease involving iliac vessel or femoral artery may relapse, despite thenegative surgical margin, given that the vessels are in the proximity of aorta (Susaengrat etal., 2019).

Serum β-d-glucan and P. insidiosum-specific antibody are potential biomarkers ofvascular pythiosis after treatment initiation (Worasilchai et al., 2018; Susaengrat et al.,2019). Serum β-d-glucan declined three months after surgery and became undetectableamong survivors (Worasilchai et al., 2018). Persistently elevated serum β-d-glucan at twoweeks after surgery should prompt an evaluation for residual disease (Worasilchai etal., 2018). Furthermore, an elevated trend of β-d-glucan from baseline should promptan investigation and treatment for disease relapse (Susaengrat et al., 2019). Deathpatients had statistically significant higher levels of serum β-d-glucan, compared withsurvivors (Worasilchai et al., 2018).

A robust host immune response to PIA vaccine is demonstrated by P. insidiosum-specific antibody titer level. A high level of P. insidiosum-specific antibody measured byELISA after PIA vaccine administration shows a statistically significant association withsurvival (Worasilchai et al., 2018).

Ocular pythiosisSurgical interventionEarly therapeutic penetrating keratoplasty with at least one mm clear margin is consideredthe gold standard for eradicating Pythium keratitis. Radiating reticular pattern must beincluded within the keratoplasty. Complete elimination of the exudate from the anteriorchamber prevents recurrence of infection. Patients with global salvage underwent thefirst surgical excision earlier than patients who eventually lost their eyes (Permpalunget al., 2019). However, in eyes with very large or peripheral lesions extending up to thelimbus, the outcome of therapeutic penetrating keratoplasty may be poor with a high riskof recurrence, because the surgical margin may not be free of infection. Adjunct therapyis therefore recommended in such cases to reduce the risk of recurrence (Agarwal et al.,

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2019; Agarwal et al., 2018). Application of single freeze-thaw cryotherapy on the host at thegraft-host junction or at the limbus using 2 mm-tip retinal cryoprobe for 7–8 s has beenused successfully to prevent recurrence (Agarwal et al., 2018). Additionally, in patients withscleral involvement, 99.9% absolute alcohol should be applied over and beyond the area ofcryotherapy application extending to the posterior edge of infected sclera for 20 s to reducethe potential detrimental effects of multiple rows of cryotherapy (Agarwal et al., 2018). Toavoid delays in surgery, glycerin-preserved corneal grafts or scleral grafts are alternativesto corneal grafts when corneal donor tissues are not available (Thanathanee et al., 2013).Evisceration or enucleation may be required in cases of extensive lesions involving largearea of sclera or endophthalmitis (Lekhanont et al., 2009; Neufeld et al., 2018).

Antimicrobial agentsAsmentioned earlier (see vascular pythiosis section), P. insidiosum is generally refractory toantifungals, which reflects the failure of medical treatment in majority of cases. However,few cases were resolved with topical 1% itraconazole and only one eye of a patient withbilateral keratitis responded to medical therapy (0.15% amphotericin B, 2% ketoconazole,1% voriconazole, and oral terbinafine) (Anutarapongpan et al., 2018; Hasika et al., 2019).

Despite the lack of management strategies for Pythium keratitis, recent advances intherapeutics are encouraging. Several studies showed in vitro susceptibility of variousstrains of P. insidiosum to antibacterial agents that inhibit protein synthesis includingtigecycline, macrolides, tetracyclines, and linezolid, either as monotherapy or as adjuncttherapy to antifungal agents (Mahl et al., 2012; Jesus et al., 2014; Jesus et al., 2016; Loretoet al., 2014). A large ocular series showed in vitro activity of tigecycline, mupirocin, andminocycline against Indian P. insidiosum strains (Bagga et al., 2018). The first patientwith presumptive Pythium keratitis was successfully managed non-surgically with a tripleregimen, consisting of topical 0.2% linezolid every hour, topical 1% azithromycin every2 h, and oral azithromycin 500 mg once daily for 3 days a week (Ramappa et al., 2017).One presumptive case and two confirmed cases of Pythium keratitis were successfullytreated with similar regimens, including a combination of topical and oral azithromycinand topical voriconazole, a combination of topical minocycline and chloramphenicoland oral linezolid, and a combination of oral doxycycline and clindamycin and topicalazithromycin, respectively (Chatterjee & Agrawal, 2018; Maeno et al., 2019; Bernheim etal., 2019). Intracameral injection and oral minocycline have also been used along withrepeated keratoplasty to successfully treat a patient with recurrent infection after thesecond corneal transplant (Ros Castellar et al., 2017). A favorable, but not statisticallysignificant, response of Pythium keratitis to a triple combination of topical linezolid andtopical and oral azithromycin was found in a large pilot series of 18 patients (Bagga et al.,2018). The percentage of success and failure in smaller lesions (≤ 6mm) was approximatelyequal and there was 100% failure in eyes with larger lesions (>6 mm) (Bagga et al., 2018).Furthermore, all patients in another series failed to respond and/or worsened with thetriple regimen (Agarwal et al., 2019). Additional clinical trials are needed to investigate thetrue efficacy of these antibiotics, which have a common mechanism of action —inhibition

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of protein synthesis. Thus, treatment may be considered an adjuvant therapy after surgicalexcision.

ImmunotherapyFor some patients with ocular pythiosis, PIA has been used together with other therapeuticmodalities as compassionate therapy (Lekhanont et al., 2009; Permpalung et al., 2015;Permpalung et al., 2019; Thanathanee et al., 2013). Three patients received subcutaneousadministration of PIA to prevent post-keratoplasty recurrence in corneal grafts (Lekhanontet al., 2009; Thanathanee et al., 2013). Although no recurrence was seen in two of threepatients after re-graft, combined with three doses of vaccine, this may be attributed tokeratoplasty with a wide surgical excision. The other two series reported the use of PIAat diagnosis and showed that 43–47% of patients needed evisceration/enucleation to becured of the disease (Permpalung et al., 2015; Permpalung et al., 2019). It is interesting tonote that the use of PIA has been reported solely from Thailand. To be declared effective,larger, well-designed studies on the efficacy and safety of PIA are necessary.

Follow-up and monitoringSlit-lamp examination of Pythium keratitis patients may demonstrate a remarkableincrease in corneal infiltration in a day (Lekhanont et al., 2009). Therefore, daily clinicalmonitoring is highly recommended. Recurrence after therapeutic penetrating keratoplastycould be as high as 45–75% of cases (Anutarapongpan et al., 2018; Agarwal et al., 2019;Thanathanee et al., 2013; Agarwal et al., 2018), and occurs within the first two weeks aftersurgery (Lekhanont et al., 2009; Thanathanee et al., 2013). The graft-host junction andthe anterior chamber should be carefully inspected for new infiltration or newly formedwhite exudate inside the chamber (Agarwal et al., 2019). If the warning signs are present,prompt re-grafting and adjunct therapies should be considered to avoid eye loss. Extensiveinfection involving the sclera and extraocular muscles could potentially lead to cavernoussinus thrombophlebitis (Rathi et al., 2018; Chitasombat et al., 2018b). Thus, imaging studyof brain and orbit should be considered to determine disease extension and treatmentplanning.

Clinical outcomesVascular pythiosisThe mortality rate of vascular pythiosis ranged between 31% and 100% (Krajaejun et al.,2006b; Permpalung et al., 2015; Chitasombat et al., 2018a; Sermsathanasawadi et al., 2016).The main predictor of survival was the microscopic demonstration of P. insidiosum-free surgical margin (vascular and soft tissue) (Sermsathanasawadi et al., 2016). Diseaserecognition is an importantmeasure for pre-surgical diagnosis, adequate surgery, and tissuediagnosis (Chitasombat et al., 2018a). Delay in diagnosis and surgical treatment contributedto advanced disease which was beyond surgical cure (Permpalung et al., 2015; Reanpanget al., 2015; Sermsathanasawadi et al., 2016). A recent prospective cohort study exploredthe use of new monitoring tools, including serum β-d-glucan and P. insidiosum-specificantibody, and showed that the mortality rate reduced to 10%, given that most patientshad free surgical margin (Worasilchai et al., 2018). Patients with unresectable disease

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involving the common iliac artery and aorta often did not survive (Krajaejun et al., 2006b;Permpalung et al., 2015; Sermsathanasawadi et al., 2016). There are few reports of patientswith suprainguinal vascular pythiosis who survived after aggressive surgical eradication,i.e., amputation and surgical removal of all infected arteries, extending to the common iliacartery, in conjunction with antifungal agent, and PIA immunotherapy (Hahtapornsawan etal., 2014). Recently, new therapeutic modalities comprising a combination of antifungals,adjunct antimicrobials and PIA vaccine, along with surgery successfully stabilized disease intwo patients up to 64 weeks of follow-up (Susaengrat et al., 2019). Overall survival dependson several factors; site of vascular involvement, duration of symptoms to the first surgery,definitive surgery, negative surgical margin, underlying disease, immune response to PIAvaccine.

Ocular pythiosisIn the past, up to 90% of the cases required evisceration or enucleation (Krajaejun etal., 2004; Kunavisarut, Nimvorapan & Methasiri, 2003). Recently, a large case series of 46eyes with Pythium keratitis demonstrated that the rate of recurrence after penetratingkeratoplasty was 55% and the evisceration rate was reduced to 15% of the cases (Agarwalet al., 2019). None of the cases recurred following therapeutic keratoplasty with adjunctprocedures and enucleation was not needed (Agarwal et al., 2019). Available data show thatexperienced clinicians who promptly perform surgery and provide adjunct measuresincluding cryotherapy and absolute alcohol treatment, enable favorable treatmentoutcome.

CONCLUSIONSHuman pythiosis is a life-threatening condition with high morbidity and mortality.Vascular and ocular pythiosis are common clinical manifestations. Early diagnosis andtimely intervention are the keys to an optimal outcome. Although diligent records of patientmedical history and physical examination provide diagnostic clues on ‘‘pythiosis’’, definitivedisease diagnosis requires laboratory testing. In addition to traditional microbiologicalmethods, diagnostic assays (i.e., serological, molecular, and proteomic tests) have beendeveloped to aid diagnosis of pythiosis. Selection of the diagnostic tests relies on assayavailability, detection efficiency, and experience of laboratory personnel. Regarding themanagement of vascular pythiosis, surgical intervention that achieves a Pythium-freemargin of the affected tissue, in combination with the administration of antifungaldrugs and PIA, remain the recommended treatment. Use of adjunct antimicrobials holdsconsiderable promise in mitigating disease relapse. During treatment, clinical assessmentof the vascular pythiosis patient should be performed daily. Ocular pythiosis is a criticalophthalmological condition where a high degree of suspicion and precise recognition ofthe typical clinical features of ocular pythiosis are warranted for early diagnosis. While therole of therapy for ocular pythiosis (i.e., antimicrobial drugs and PIA) remains uncertain,early therapeutic penetrating keratoplasty with clear surgical margins is the gold standardfor achieving global salvage. To prevent recurrence after eye surgery, adjunct strategiesincluding perioperative cryotherapy and absolute alcohol application may be beneficial.

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ACKNOWLEDGEMENTSWe thank Professor Boonmee Sathapatayavongs for her kind support.

ADDITIONAL INFORMATION AND DECLARATIONS

FundingThis study was supported by the Faculty of Medicine, Ramathibodi Hospital, MahidolUniversity (grant number, CF61007), and Thailand Research Fund (grant number,RSA6280092). The funders had no role in study design, data collection and analysis,decision to publish, or preparation of the manuscript.

Grant DisclosuresThe following grant information was disclosed by the authors:Faculty of Medicine, Ramathibodi Hospital, Mahidol University: CF61007.Thailand Research Fund: RSA6280092.

Competing InterestsThe authors declare there are no competing interests.

Author Contributions• Maria Nina Chitasombat, Passara Jongkhajornpong, Kaevalin Lekhanont andTheerapong Krajaejun conceived and designed the experiments, analyzed the data,prepared figures and/or tables, authored or reviewed drafts of the paper, and approvedthe final draft.

EthicsThe following information was supplied relating to ethical approvals (i.e., approving bodyand any reference numbers):

The Committee for Research, Faculty of Medicine Ramathibodi Hospital MahidolUniversity granted Ethical approval to carry out the study within its facilities(MURA2019/740; MURA2019/1228).

Data AvailabilityThe following information was supplied regarding data availability:

The study (review article) generated no data nor code.

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